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Comprehensive diagnosis of Rett’s syndrome
relying on genetic, epigenetic and expression
evidence of deficiency of the methyl-CpG-binding
protein 2 gene: study of a cohort of Israeli patients
Y Petel-Galil, B Benteer, Y P Galil, B B Zeev, I Greenbaum, M Vecsler, B Goldman,
H Lohi, B A Minassian and E Gak
J. Med. Genet. 2006;43;56doi:10.1136/jmg.2006.041285
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ELECTRONIC LETTER
Comprehensive diagnosis of Rett’s syndrome relying on
genetic, epigenetic and expression evidence of deficiency of
the methyl-CpG-binding protein 2 gene: study of a cohort of
Israeli patients
Y Petel-Galil, B Benteev, Y P Galil, B B Zeev, I Greenbaum, M Vecsler, B Goldman, H Lohi,
B A Minassian, E Gak
...............................................................................................................................
J Med Genet 2006;43:e56 (http://www.jmedgenet.com/cgi/content/full/43/12/e56). doi: 10.1136/jmg.2006.041285
Background: Despite advances in the characterisation of
mutations in the MECP2-coding region, a small proportion of
classic RTT cases remain without recognisable mutations.
Objective and methods: To identify previously unknown
mutations, a quantitative assay was established, providing
estimates of MECP2_e1 and MECP2_e2 expression levels in
peripheral blood. A systematic analysis of an Israeli cohort of
82 patients with classic and atypical RTT is presented,
including sequence analysis of the MECP2-coding region,
MLPA, XCI and quantitative expression assays.
Results and conclusion: A novel mis-sense mutation at ca
453CRT (pD151E), resulting in a change of a conserved
residue at the methyl-binding domain, and a rare GT
deletion of intron 1 donor splice site are reported. It is
shown that various MECP2 mutations had distinct effects on
MECP2 expression levels in peripheral blood. The most
significant (p,0.001) reduction in the expression of both
MECP2 isoforms was related to the presence of the intron 1
donor splice-site mutation. Using quantitative expression
assays, it was shown that several patients with classic and
atypical RTT with no mutation findings had significantly lower
MECP2 expression levels. Further research on these patients
may disclose still elusive non-coding regulatory MECP2
mutations.
R
ett syndrome (RTT; OMIM 312750) is a neurodevelopmental disorder characterised by cognitive impairment,
communication dysfunction, microcephaly, growth failure and stereotypic hand movements. RTT primarily affects
females and is sporadic in most cases, with a worldwide
incidence of 1/10 000–15 000 female births.1 In most cases,
symptoms of RTT become apparent during the initial years of
life, when patients fail to achieve or regress from the
expected developmental milestones and become engaged in
characteristic hand-wringing motions. Most of the patients in
addition have breathing abnormalities, about a half of them
develop seizures and a third develop scoliosis. By age 4–
7 years, most patients develop gross cognitive and motor
impairments, leading to profound lifelong hypoactivity.2 3 The
diagnosis of RTT is essentially clinical and is based on
fulfilment of consensus criteria for both the classic and
atypical forms; atypical RTT includes forme fruste, preserved
speech variant (PSV), late regression variant, and congenital
and early seizure onset variants.4 RTT has been associated
with mutations in the methyl-CpG-binding protein 2
(MECP2) gene,5 and additional clinical phenotypes have been
Key points
N
N
N
This study introduces a novel assay providing quantitative estimates of expression levels of both MECP2_e1
and MECP_e2 isoforms in peripheral blood that could
support the diagnosis of patients with Rett syndrome
(RTT) without recognisable MECP2 mutations.
We implemented these quantitative expression assays
in a systematic analysis of an Israeli cohort of RTT
patients together with analyses of MECP2 coding
sequence, MLPA and XCI.
We show that quantitative expression assays distinguish between various MECP2 mutations, with significantly reduced peripheral MECP2 expression in
cases with the intron 1 donor splice-site mutation and
normal expression in cases with mis-sense mutations
and small in-frame deletions. In addition, we show
several classical and atypical patients with no previous
mutation findings that have lower peripheral MECP2
expression levels, which suggest the possibility of yet
unidentified MECP2 mutations.
included on genetic basis, among them Angelman-like
phenotype and familial X-linked metal retardation, and
autism in males (reviewed by Naidu et al6).
From the genetic standpoint, RTT is an X-linked dominant
disorder caused by defects in the MECP2 gene on chromosome Xq28. MECP2 mutations were detected in .85% of
patients with classic RTT, most of which (about 70%) involve
CRT transitions at specific CpG hot spots in exons 3 and 4;
an additional 10% involve small deletions at the 39 end of the
gene.7 After the finding of the novel MECP2_e1 transcription
isoform,8 9 mutation analysis of MECP2 has been extended to
include exon 1 that was previously considered untranslated.
The use of novel molecular technologies, such as denaturing
high-performance liquid chromatography, multiple ligationdependent probe amplification (MLPA) and genomic-based
quantitative polymerase chain reaction (PCR),10–12 has
increased the overall detection rate of MECP2 mutations in
patients with classic RTT to nearly 90%. An additional 15% of
Abbreviations: MECP2, methyl-CpG-binding protein 2; MLPA, multiple
ligation-dependent probe amplification; PCR, polymerase chain
reaction; PSV, preserved speech variant; QtPCR, quantitative PCR
reaction; RTT, Rett’s syndrome; UTR, untranslated region; XCI, X
chromosome inactivation
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atypical RTT cases, particularly early seizure onset variants,
have been resolved by the recent findings of mutations in the
X-linked cyclin-dependent kinase-like 5 gene.13 14 However,
despite these advances in the molecular diagnosis of RTT, the
remaining 10% of patients with classic RTT and .50% of
those with atypical RTT continue to pose a diagnostic
dilemma. As MECP2 is located on the X chromosome, another
modulator of the RTT phenotype is the X chromosome
inactivation (XCI) pattern. Preferential activation of the
normal allele is one of the possible explanations of asymptomatic female carriers of RTT-causing mutations or those
having minor learning disabilities.15
The conventional means of obtaining molecular evidence
of RTT has been based, thus far, on sequencing of the MECP2coding region and exon–intron junctions. At the same time,
mutations in the regulatory elements of MECP2, which
ultimately affect MECP2 expression levels, have been omitted
from this analysis. We considered an additional approach to
the molecular diagnosis of RTT on the basis of the analysis of
MECP2 expression levels in peripheral blood of patients. We
developed a rapid and direct assay providing quantitative
estimates of expression levels of both MECP2_e1 and
MECP2_e2 transcription isoforms in peripheral blood, using
real-time PCR and fluorescent-labelled isoform-specific
TaqMan probes. This approach could be potentially diagnostic in patients in whom no MECP2-coding mutations have
been previously detected. Accordingly, we preferentially
implemented MECP2 expression assays in a group of Israeli
patients and some patients referred by our US and Canadian
colleagues, in whom no MECP2 mutations had been detected.
In addition, MECP2 expression assays were also carried out
in some representative cases with MECP2 mis-sense and
nonsense mutations, C-terminal deletions and large rearrangements in the MECP2 gene region.
METHODS
Patients and controls
The Israeli RTT cohort included 82 patients of whom 52 were
diagnosed with classic and 17 with atypical RTT, including 4
with PSV, 4 with congenital variants, 6 with early seizure
onset variants, 2 with forme fruste variants and 1 male
variant, using clinical criteria established by Hagberg et al.4
The cohort also included four females with Angelman-like
features and nine patients with diagnoses reminiscent of
RTT, including seven females with autism spectrum disorder
and two males with congenital severe encephalopathy. All
the patients were classified by the same paediatric neurologist at the Neuropediatric Clinic at the Sheba Medical Center,
Tel Hashomer, Israel, where they continue to attend for
clinical follow-up. The age of patients was in the range 2–
40 years. Molecular diagnosis of RTT was carried out as a part
of an ongoing study approved by the institutional review
board or as an officially authorised diagnosis provided by
health insurance. Patients included in the expression studies
were recruited after informed consent of the parents
approved by the institutional review board. This group
included patients with classic (C1–C5) and atypical (A1–
A7) RTT. Patients C1–C5 and A1, A3 and A4 were recruited
from the Israeli cohort, patient A5 was referred from the
Hospital for Sick Children, Toronto, Ontario, Canada, and
patients A2, A6 and A7 were referred from the Rett Syndrome
Research Foundation, Cincinnati, Ohio, USA. As regards the
diagnoses of patients with atypical RTT, A1 and A2 had
congenital variants, and A3 and A4 had early seizure onset
variants, but A5, A6 and A7 had no specific diagnoses other
than atypical RTT. In addition, a sample from an atypical
patient with partial deletion of exon 4 (del exon 4a) was
provided by Dr Jane Hickey via the Rett Syndrome Research
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Petel-Galil, Benteev, Galil, et al
Foundation. Expression studies also included 12 samples
from healthy female volunteers (F1–F12) aged 17–40 years.
DNA and RNA extractions
Peripheral blood samples collected in tubes containing EDTA
anticoagulant were used for the extraction of genomic DNA
by the Puregene kit and total RNA by the Versagene kit (both
by Gentra, Minneapolis, MN, USA). Additional blood
samples were transferred from Canada and the US in
Paxgene tubes (PreAnalytix, Hombrechtikan, Switzerland)
and RNA was extracted by the same method. The yield and
purity of the DNA and RNA extractions were determined
according to the optical density ratio 260/280 nm using a
spectrophotometer (GeneQuant, Cambridge, UK). RNA samples were subjected to DNAse I treatment included in the
Versagene kit; 10–20 units of protector RNAse inhibitor
(Roche Applied Science, Penzburg, Germany) were added
and the samples were stored at 220˚C.
MECP2 mutation analysis
PCR fragments spanning the MECP2 promoter region (1.2 kb
upstream of exon 1) and the entire coding region (exons 1–4)
were generated using seven primer pairs (sequences available
on request). PCR fragments were purified on silica gel spin
columns (Bioneer, Daejon, Korea) and analysed on an ABI
Prism 3100 AVANT Genetic Analyzer (Applied Biosystems,
Foster City, CA, USA). The presence of mutations was
confirmed by replicate sequencing of independently generated PCR products.
Multiplex ligation-dependent probe amplification
Large deletions spanning MECP2 exons 1–4 were detected
using the MLPA-P015 probe (MRC, Amsterdam, The
Netherlands) according to the manufacturer’s protocol.
MLPA products were separated and identified using the
ABI Prism 3100 Genetic Analyzer using Genescan ROX-500
standards and Genescan software (both by Applied
Biosystems). Reproducible deviation of .30% of relative
peak area as compared with the normal control sample
derived from two independent experiments was considered
conclusive.
X chromosome inactivation
The XCI pattern in peripheral blood was analysed using the
human androgen receptor assay.16 Briefly, genomic DNA was
digested with the methylation-sensitive enzyme HpaII for
preferential cleavage of the unmethylated-active alleles.
Digested and undigested DNA samples were subjected to
PCR of the human androgen receptor gene, including the
highly polymorphic trinucleotide repeat region. PCR products
were separated using the ABI Prism 3100 Genetic Analyzer,
calculating the peak areas of the undigested and digested
alleles using Genescan ROX-500 standards and Genescan
software. Estimation of XCI included correction for the large
digested allele peak area considering the ratio between peak
areas of the small and the large undigested alleles. Skewed
XCI was considered to be significant for ratios >75%,
whereas reported values were derived from two independent
and reproducible experiments (,10% difference). The parental origin of the preferentially activated chromosome was
further determined by comparing the profiles of the patient’s
and the parental samples.
cDNA synthesis
Double-stranded cDNA was generated from 500 ng of total
RNA in the presence of p[dN]6 random primer hexamers
(Roche Applied Science), using an Omniscript RT kit (Qiagen
Hilden, Germany) according to the manufacturer’s protocol.
Reactions were carried out in a total volume of 40 ml.
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Expression of MECP2 in peripheral blood
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TaqMan reactions
Relative expression levels of MECP2_e1 and MECP2_e2
transcription isoforms were determined using primers
designed by Primer Express software, TaqMan probes and
PCR kit (all from Applied Biosystems). The MECP2_e1 assay,
designed by us, included forward primer from exon 1 (59CGG AGG AGG AGG AGG A) and reverse primer from exon 3
(59-GGA GGT CCT GGT CTT CTG ACT T), producing a 63-bp
amplicon, and the TaqMan probe for the exon 1–3 junction
(gene accession BX538060). The MECP2_e2 commercial
assay included the TaqMan probe for the exon 2–3 junction.
Both MECP2 probes contained 59-6-carboxyfluorescein fluorophore reporter and 39-non-fluorescent quencher. The
commercial RNAseP kit consisting of RNAseP-specific primers and VIC-labelled TaqMan probe was used as an internal
reference, enabling multiplexing of MECP2 and RNAseP
assays. Separate pre-runs of varying primer concentrations
were carried out to obtain the highest intensity and
specificity of the fluorescent reporter signal. The results were
validated in separate reactions including the commercial 6carboxyfluorescein-labelled ornithine decarboxylase 1 probe
and primers as another reference. Quantitative PCR reactions
(QtPCRs) were carried out in a volume of 20 ml in 96-well
optical plates using a common Mastermix including 26
TaqMan Universal PCR Mastermix, 900 nM MECP2_e1 or
MECP2_e2 primers, 250 nM MECP2_e1 or MECP2_e2 probe
and 206 RNAseP kit, and high-performance liquid chromatography-pure water. Aliquots of 2 ml of each cDNA species
were dispensed in three wells for triplicate reactions.
Reactions were carried out on ABI Prism 7000 (Applied
Biosystems) under uniform conditions, including a pre-run at
50˚C for 2 min and 95˚C for 10 min, and 40 cycles at 95˚C for
15 s and at 60˚C for 1 min. Each reaction plate included
triplicate samples of a normal control pool constructed from
4–5 cDNAs generated from normal females (fig 1). Each plate
also included reactions with no-template control and RNA
template (RT) for monitoring of the background signal and
DNA contamination, respectively.
Quantitative analysis
Data were evaluated using the ABI Prism 7000, comparing
the levels of MECP2_e1 and MECP2_e2 transcripts with
those of the RNAseP internal reference gene. In most cases,
the threshold of QtPCRs was automatically set at 10 standard
deviations (SD) above the mean baseline emission,
_
representing the background signal, and the specific reaction
cycle threshold (Ct) number was determined relative to this
threshold. Only samples with at least three QtPCR results
(triplicates) were included in the data analysis. The comparative (ddCt) method was used to calculate the relative
transcript number,17 previously verifying that QtPCR efficiencies of the target and reference genes were almost equal.
Efficiencies of MECP2_e1, MECP2_e2 and RNAseP reactions
were determined from standard curves generated by serial
twofold dilutions of the cDNA sample and estimation of the
cycle threshold at each dilution (fig 2). Reaction efficiencies,
calculated as E (efficiency) = 1021/slope, yielded values of
1.84, 1.86 and 1.89 for MECP2_e1, MECP2_e2 and RNAseP,
respectively, which could be considered approximately equal
(standard error ,0.02). Relative transcript number in the
patient samples was determined according to the ddCt
method comparing the patient’s sample and the pooled
sample of normal female controls using the following
equations:
dCt = CtMECP2–CtRNAseP and ddCt = dCtpatient–dCtcontrol
pool
and on inclusion of a correction for mean PCR efficiency,
MECP2_e1 transcript number = 1.862(ddCt),
MECP2_e2 transcript number = 1.842(ddCt)
Statistical methods
For the purpose of statistical analyses of MECP2_e1 and
MECP2_e2 expression data, patients were grouped into four
groups according to the MECP2 mutation type (I, splice-site
mutations; II, large deletions; III, nonsense and frame-shift
mutations; and IV, mis-sense and in-frame mutations). The
individual mean MECP2_e1 and MECP2_e2 levels in the
patient groups and those in the normal control group were
compared using analysis of variance with Bonferroni correction for multiple comparisons. Correlation analysis between
the levels of both MECP2 isoforms was carried out using the
Spearman correlation procedure. Multinomial (polytomous)
regression was applied to predict the relatedness of patients
with no known MECP2 mutation to mutation groups I–IV,
while combining groups I and II. All data were analysed
using SAS V.9.1.3 for Unix via procedures MIXED and
LOGISTIC.
_
Figure 1 Quantitative analysis of peripheral methyl-CpG-binding protein 2 (MECP2)_e1 and MECP_e2 expression levels in healthy female controls.
Expression levels of both MECP2 isoforms in normal female control samples (F1–F12) were determined according to the ddCt method using RNAseP as
a reference gene. MECP2_e1 (grey) and MECP2_e2 (black) expression levels were normalised to the mean dCt value.
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Petel-Galil, Benteev, Galil, et al
_
_
Figure 2 Validation of amplification efficiencies of the target methyl-CpG-binding protein 2 (MECP2) gene and reference RNAseP gene transcripts.
Standard curves plotting the log of cDNA dilutions versus cycle threshold (Ct) were generated in duplicates by serial (factor 2) dilutions of the cDNA
sample. Quantitative polymerase chain reaction analyses were carried out in the presence of TaqMan probes specific to the MECP2_e1 exon 1–3
junction (white), MECP2_e2 exon 2–3 junction (grey) and exonic RNAseP (black). Efficiencies of each assay were calculated from the curve slopes
(E = 1021/slope).
RESULTS
Analyses of MECP2 mutations, MLPA and XCI
MECP2 mutation analysis of the Israeli cohort (n = 82) yielded
54 patients with MECP2 sequence variations, including 42 with
classic RTT, 4 with PSV, 2 with congenital variants, 1 with
forme fruste variant and 5 with atypical RTT. Our mutation
detection rate is 80% (42/52) as regards patients with classic
RTT. Table 1 summarises the clinical and mutation data of
patients, including XCI regarding preferential activation of the
maternal or paternal X chromosome. Apart from the known
MECP2-coding mutations and various microdeletions at the 39coding end, we identified a novel mis-sense mutation at
c453CRG (p D151E) in one patient with PSV, located at the
MECP2 methyl-binding domain. In two patients with classic
RTT, we detected a rare GT deletion in an intron 1 donor splice
site. In one patient with classic RTT, we detected changes in
both MECP2 alleles, including a 1.1-kb deletion in the intron 3–
exon 4 region and a novel maternally inherited sequence
variation at c824TRC (p V275A). Using MLPA, we detected a
large (>4.2 kb) deletion spanning parts of exon 4 and the 39
untranslated region (UTR) in one congenital variant. We also
detected four maternally inherited sequence variations
(table 1, lower panel), among which c378-19delT, c602CRT
(p A201V) and c1451GRC (p R484T) have been reported in
the International Rett Syndrome Association database as
polymorphisms, but c753CRT (p P251P) is a novel mutation.
Among the patients with MECP2 mutations, we detected
significantly skewed (>75%) XCI in 11 of 48 (23%)
informative cases, including three patients with preferential
activation of the paternal X and eight patients with preferential activation of the maternal X chromosome. Ultimately,
ten patients with classic RTT remained with no indication of
MECP2 mutation, either by sequence analysis or by MLPA. Five
of these patients were further included in quantitative
analyses of MECP2 expression in peripheral blood (C1–C5),
together with seven patients with atypical RTT with no
mutation findings (A1–A7).
Development of quantitative assay for the analysis of
MECP2 expression levels
We developed an additional strategy for the detection of
MECP2 deficiency, introducing quantitative analysis of
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MECP2 expression levels in peripheral blood, using TaqMan
probes for the target genes, MECP2_e1 and MECP2_e2, and
RNaseP as a reference gene. QtPCR efficiencies of the target
and reference genes were estimated from the cycle threshold
values obtained from serial dilutions of a cDNA sample.
Figure 2 shows that all QtPCR efficiencies were similar and
within the range 92–95% of maximal efficiency. MECP2_e2
was detected at a lower cycle threshold (difference of 4Ct),
suggesting that the levels of MECP2_e2 are ,8 times as
abundant in the peripheral blood, which is also consistent
with other reports.9 The normal range of MECP2_e1 and
MECP2_e2 expression levels in peripheral blood was estimated by the analysis of a series of normal female control
samples (F1–F12). Figure 1 shows that in most cases,
MECP2_e1 and MECP2_e2 expression levels in the normal
female controls were analogous and differed by factor of 1.5
(0.09) for MECP2_e1 and 1.7 (0.14) for MECP2_e2. Thus, in
the following expression assays we included a pooled sample
of four cDNAs from normal female controls.
Analysis of MECP2_e1 and MECP2_e2 expression
levels in patients with known mutations
To validate the consistency of quantitative expression assays
with the presence of known MECP2 mutations, we examined
the expression levels of MECP2_e1 and MECP2_e2 in the
peripheral blood of 15 such patients. Figure 3A,B shows this
analysis. Dotted lines indicate the normal range of MECP2
expression in normal female controls. Grouping the patients
into four mutation groups, including splice-site mutations,
large deletions, truncating (nonsense and frame-shift) and
non-truncating (mis-sense and in-frame) mutations, we
compared MECP2 expression levels between these groups
and the group of normal controls. We found significant
differences in MECP2 expression levels between various
mutation groups (F = 19.01, df = 4, p,0.001 for MECP2_e1;
and F = 23.01, df = 4, p,0.001 for MECP2_e2 by analysis of
variance and Bonferroni correction). The group with the
splice-site mutations had significantly lower levels of both
MECP2 isoforms than the normal controls (p,0.001 for
MECP2_e1 and p = 0.003 for MECP2_e2 by analysis of
variance and Bonferroni correction). The large deletions were
lower specifically for MECP2_e2 (p = 0.069 for MECP2_e1;
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Expression of MECP2 in peripheral blood
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Table 1 Characterisation of the Israeli cohort with Rett syndrom by analyses of mutation analysis of the methyl-CpG-binding
protein 2-coding region, multiple ligation-dependent probe amplification and X chromosome inactivation
Rett phenotype
Nucleotide position
Amino acid change
Functional domain
XCI pattern (preferentially
active X)
1 Classic
(donor 1a)
1 Classic
(donor 1b)
1 Classic
2 Classic
c 62+1delGT
Splicing alteration
Intron 1 splice donor
R*
c 62+1delGT+378-19delT
Splicing alteration
Intron 1 splice donor
S (90% paternal X)*
c 141insA
c 316CRT
p E55fs57X
p R106W
Up MBD
Up MBD
1
2
1
1
1
7
5
c
c
c
c
c
c
c
Rearrangement and p V275A TRD
p R133C
MBD
p S134C
MBD
p D151E
MBD
p T158M
MBD
p T158M
MBD
p R168X
Inter-domain
1 Classic
1 Classic
8 Classic
c 674CCRTG
c 731insC
c 763CRT
p P225R
p Q244fs258X
p R255X
TRD
TRD
TRD-NLS
1 Classic
1 Classic
2 Classic
c 775_995del221
c 806delG
c 808CRT
p A259fs266X
p G269fs288X
p R270X
TRD-NLS
TRD-NLS
TRD-NLS
1 Congenital
1 Classic
1 Classic
1 Classic, 1 FF
1 Classic
1 Classic
1 Classic
1 PSV
1 Congenital
(del exon 4b)
1 Congenital
female,
1 Rett-like male
1 Angelman like
1 Autism with
seizures
1 Autism
c
c
c
c
c
c
c
c
c
p R270fs288X
p R294X
p I303_T400del98
p R306C
p P360fs365X
p L386fs486X
p L386fs431X
p L386-S401del15insP
Large rearrangement
TRD-NLS
TRD
TRD
TRD
C-terminus
C-terminus
C-terminus
C-terminus
C-terminus_39 UTR
S (80% maternal X)
2 S (90%* and 80%
maternal X)
R
4R
R
NI
R*
6 R, 1 NI
3 R, 1 S (90% maternal X),
1 NI
R
R*
6 R*, 1 S (80% maternal X),
1 NI
R*
S (80% maternal X)
2 S (75% maternal X and
75% paternal X*)
R
R
R*
2R
R
R
R*
R*
S (80% paternal X*)
c 378-19delT
Probably none
Intron 3
NI (female*)
c 602CRT
c 753CRT
p A201V
p P251P
Interdomain
TRD
R
S (85% maternal X)
c 1451GCRTC
p R484T
C-terminus
R
Classic
PSV, 2 classic
Classic
PSV
Classic
Classic
Classic
378-219_1164del1018+824TRC
397CRT
401CRG
453CRG
473CRT+378-19delT
473CRT
502CRT
808delC
880CRT
908_1201del294
916CRT
1080 _1161del82
1157_1197del41
1157_1327del171
1157_1198del42
TRD_39 UTR >4.2 kb del
FF, forme fruste; MBD, methyl-binding domain; NI, non-informative case; NLS, nuclear localisation signal; PSV, preserved speech variant; R, random X
chromosome inactivation; S, skewed X chromosome inactivation; TRD, transcriptional repression domain; UTR, untranslated region; XCI, X chromosome
inactivation.
The table includes data on RTT subtype, MECP2 mutation type and position according to the accepted nomenclature, localisation within the MECP2 functional
domain and pattern of XCI considering preferential activation of the maternal or paternal X chromosome. Mutations were confirmed with the International Rett
Syndrome Association database. Cases reported in the lower panel are with inherited MECP2 polymorphisms or variations with unknown significance.
*Patients were included in quantitative expression assays.
p,0.001 for MECP2_e2); by contrast, the truncating mutations were lower for both isoforms (p,0.001 for MECP2_e1;
p = 0.002 for MECP2_e2). The non-truncating mutations
were similar to the normal controls for both MECP2 isoforms
(p = 1.000 for MECP2_e1; p = 0.161 for MECP2_e2). The
overall expression levels of MECP2_e1 and MECP2_e2 were
significantly correlated (Spearman r = 0.76; p,0.001).
Quantitative expression analysis of patients negative
for MECP2 mutations
To obtain evidence of MECP2 mutations in patients with no
previous mutation findings, we included 12 such patients in
quantitative expression assays, five with classic (C1–C5) and
seven with atypical (A1–A7) RTT. We took special care of the
transport of blood samples from Canada and the US to ensure
that the authentic RNA concentration was maintained (see
Methods). Figure 3A,B shows our findings in this group. We
correlated the patients with no mutations to the previous
groups with mutations, while combining the groups with the
splice-site mutations (group I) and the large deletions (group
II) on the basis of relative similarity of small groups
(p = 0.135 for MECP2_e1 and p = 1.000 for MECP2_e2 by
analysis of variance and Bonferroni correction). This analysis
suggested that patients C2, C4, C5, A2, A5 and A7 may belong
to mutation groups I and II (predicted probabilities to belong
to these groups are p = 0.999, 0.999, 1.000, 0.919, 0.993 and
1.000, respectively, including MECP2_e1 and MECP2_e2 in
polytomous regression analysis). Patients A3 and A6 were
related to group III with the truncating mutations (p = 0.979
and 0.919, respectively). Patient C3 was related to the group
with normal expression levels by both MECP2 isoforms
(p = 1.000). Patients C1, A1 and A4 had discordant levels of
MECP2_e1 and MECP2_e2; in particular one of the isoforms
was overexpressed.
Effect of XCI on quantitative expression of MECP2 in
peripheral blood
Among the 27 patients included in the quantitative expression assays, seven were detected with considerably skewed
XCI. Preferential activation of the paternal X chromosome
was detected in patients with the splice-site mutation (donor
1a), the large deletion (del exon 4b), p R270X (XCI data in
table 1) and patient C2 (80% XCI). Preferential activation of
the maternal X chromosome was found in patients with p
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6 of 8
Petel-Galil, Benteev, Galil, et al
Figure 3 Quantitative analysis of methyl-CpG-binding protein 2 (MeCP2)_e1 and MeCP2_e2 expression levels in blood in patients with Rett
syndrome (RTT), using RNAseP as the reference gene. The charts include patients with RTT with known MECP2 mutations and X chromosome
inactivation status (table 1), patients with 62+1delGT deletion of intron 1 donor splice site (donors 1a and 1b), deletions spanning exon 4 and the 39
untranslated region (del exons 4a and 4b), nonsense mutations (R255X, R270X and R294X), frame-shift deletions (Q244fs, A259fs, P360fs, L386fs),
mis-sense mutations (R106W, T158M) and in-frame deletions (I303del98, L386del15). In addition, the charts include patients with classic RTT (C1–C5)
and atypical RTT (A1–A7) with no MECP2 mutation findings. Expression levels of (A) MECP2_e1 and (B) MECP2_e2 isoforms were determined
according to E2ddCt using RNAseP as the reference gene. Dotted lines indicate the normal expression range.
R255X and p R106W (table 1) and patient C4 (80% XCI).
Patient C1 was non-informative and other patients had
random XCI. We observed interindividual differences
between the patients with the same splice-site mutation
(donors 1a and 1b), as donor 1b with preferential expression
of the paternal X chromosome had lower MECP2_e1 and
MECP2_e2 expression levels than donor 1a with random XCI
(fig 3A,B). Also, in the two patients with two similar
nonsense mutations, p R270X with preferential activation
of the paternal X had lower MECP2_e1 and MECP2_e2
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expression levels, whereas p R255X with activation of the
maternal X chromosome had normal levels of both MECP2
isoforms.
DISCUSSION
This study presents the result of molecular diagnosis of an
Israeli cohort of patients with RTT, including various analyses
of the MECP2 gene at the genomic and expression levels. The
present cohort included 82 unrelated patients with RTT with
classic (n = 52), atypical17 and related phenotypes.13 We were
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Expression of MECP2 in peripheral blood
able to provide molecular diagnosis in 80% of classic RTT
cases and all PSV variants. The other atypical forms including
congenital and forme fruste variants were only partially
resolved. No mutations were detected in variants with early
seizure onset. Consistent with previous estimates,7 the
recurrent hot-spot mutations comprised about 65% and 39end microdeletions comprised an additional 12% of the
disease-causing mutations in our cohort. We here report a
novel mis-sense mutation c453CRG (p D151E) resulting in a
change of a conserved residue at the methyl-binding
domain.18 We also report two maternally inherited polymorphisms, c824TRC (p V275A) and c753CRT (p P251P),
the first present in conjunction with a large MECP2 deletion
and the second located at the position where another nonpathogenic mis-sense variation was previously reported.19
Another rare GT deletion of an intron 1 donor splice site,
which has been reported in two other studies,20 21 was
detected in two patients from our cohort.
The question of genotype–phenotype correlation has been
answered in a further analysis of patients, aged .5 years,
with mis-sense 17 patients and early-truncating mutations
(22), whose clinical diagnoses were scored according to the
severity scale adopted from Huppke et al.22 Consistent with a
previously suggested notion,23 24 mis-sense mutations were
associated with milder RTT phenotypes (p = 0.002 by student
t-test). Other functional analyses of MECP2 mis-sense
mutations suggest that clinical severity is also dependent
on the location of the mutation within the particular MECP2
functional domain.25 The contribution of XCI to RTT clinical
phenotype is suggested by the finding of a higher proportion
of cases with maternally skewed XCI in our cohort, which is
similar to observations in other collections and mouse
models.26 27 Although skewed XCI in peripheral blood does
not necessarily reflect XCI patterns in the brain, our results
may suggest that preferential activation of the maternal X,
which in most patients harbours the normal MECP2,28
accounts for milder RTT phenotypes.
The major advance proposed in this study is the examination of expression levels of both MECP2 isoforms in
peripheral blood samples from patients with RTT. The
question whether quantification of MECP2 expression in
vivo might provide yet another molecular indicator of MECP2
deficiency has been considered by systematic analysis of
normal females as well as patients with classic and atypical
RTT with known MECP2 mutations and patients with no
mutation findings. Using the quantitative assay that determined the relative expression levels of MECP2_e1 and
MECP2_e2 isoforms, we showed that the expression levels
of both MECP2 isoforms in normal females fall within a
relatively narrow range. Under these conditions, patients
with RTT with certain MECP2 mutations were situated below
the normal range (fig 3A,B). We showed distinct effects of
various mutations on MECP2 expression levels, and the most
marked reduction in the expression levels of both MECP2
isoforms was detected in two patients with ca 62+1delGT
deletion of an intron 1 donor splice-site. A previous study
showed that this mutation causes a complete skipping of
exon 1, resulting in elimination of the MECP2 message and
protein in lymphoblast clones of patients with RTT.29 Large
deletions of the MECP2 39-coding region and 39 UTR also
showed lower expression levels, probably as a result of the
impairment of 39-end regulatory sequences important for
mRNA processing, polyadenylation and stability.30 Unlike
other mutation types, the effect of large deletions was more
evident in the expression levels of MECP2_e2. The functional
implications of these findings are not clear and need further
replication and analysis. Truncating mutations resulting
from early nonsense or frame-shift deletions showed lower
MECP2 expression levels that could be attributed to the
7 of 8
nonsense-mediated mRNA decay mechanism.31 The missense mutations and in-frame deletions retained normal
MECP2 expression levels, thus supporting the notion that
these mutations affect the MECP2 structure and function
rather than the level of MECP2 transcripts. Also, the novel p
D151E variation had normal MECP2_e1 and MECP2_e2
expression levels using ornithine decarboxylase 1 as an
alternative reference gene (data not shown). Another study
that examined the in vivo effects of various MECP2-coding
mutations showed that there were distinctive profiles of
histone modifications in peripheral cells in patients with RTT,
which may be relevant to neurological dysfunction in RTT.32
The interindividual differences in MECP2 expression
between carriers of the same or similar mutations could be
explained by an additional effect of the XCI pattern. This was
particularly evident from the expression levels in two patients
with an identical splice-site mutation, in which the patient
with a skewed paternal XCI had lower MECP2 (donor 1b). In
the same way, maternally skewed XCI minimised the effect
of p R255X nonsense mutation and paternally skewed XCI
augmented the outcome of p R270X mutation. These findings
suggest that peripheral MECP2 expression levels reflect the
genetic and epigenetic status of the patient, and thus may be
used as yet an additional factor in RTT molecular diagnosis.
However, other factors modulating MECP2 mutation expression have been suggested and demonstrated, in particular in
males with RTT with MECP2 truncation mutations.33 The
question whether the peripheral MECP2 expression levels are
associated with phenotypic indices and prognosis of RTT
should be considered in future studies on a larger series of
patients with RTT, with comprehensive characterisation at
the clinical and molecular levels.
Ultimately, quantitative expression assays were intended
to resolve the diagnosis of patients with RTT with no previous
mutation findings. In view of a persistent lack of molecular
diagnosis in at least 10% of patients with classic RTT, we
proposed that direct estimates of the peripheral MECP2
expression levels could provide alternative indicators of the
presence of yet unknown mutations in MECP2 or other genes
that cause MECP2 deficiency. We found that in three of five
patients with classic RTT (C2, C4 and C5) and three of seven
patients with atypical RTT (A2, A5 and A7), the peripheral
MECP2 expression levels were consistent with the presence
of splice-site or deletion mutations. Our previous analysis
suggested that a lower MECP2_e2 level is specifically
associated with the presence of large deletions including
MECP2 39 UTR, whereas damage to the splice sites affects
both MECP2 isoforms. The question whether this dichotomy
is conclusive should be further investigated. We analysed
these patients by direct sequencing of candidate regions that
are highly conserved and may potentially contain regulatory
elements, including 1500 bp surrounding the putative MECP2
promoter region, and at least 500 bp upstream and downstream of the intronic boundaries, and at least 1500 bp
downstream into the 39 UTR. The major problem with this
analysis, however, is the difficulty obtaining samples of both
parents to exclude the presence of naive polymorphisms
unrelated to RTT. It is also possible that the expected defect is
located in other genes that affect MECP2 expression. Our
findings of excessive MECP2 expression in several patients
(C1, A1 and A4) are ambiguous and need further verification,
although recent studies on humans and mice have suggested
that over-expression of MECP2 is also pathological.34 35
This study essentially stems from the notion that MECP2
deficiency is the central cause of RTT. Although based on a
limited number of patients, this study suggests that almost
all patients with classic RTT can be related to MECP2
deficiency by systematic analysis of MECP2 at the genomic or
expression level. Yet, even when using such an extended
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8 of 8
approach, patient C3 had no indication of MECP2 defect in
any of the various tests applied in this study. Today, this
patient is 5 years old and is still diagnosed with classic RTT.
Although normal MECP2 expression levels do not exclude
MECP2-related disease, this case imposes some reservations
as to the exclusivity of MECP2 in RTT. The question whether
the classic RTT is a single-gene disorder remains open.
Nevertheless, our experience suggests that the inclusion of
direct MECP2 expression assays in peripheral blood in the
molecular diagnostic procedure may provide additional
information on genetic and epigenetic conditions associated
with MECP2 deficiency and a broader molecular support of
RTT diagnosis.
ACKNOWLEDGEMENTS
We thank the Israeli, the Canadian and the American families with
patients with RTT for their willingness to participate in this study and
their confidence in our work. We thank the Israeli Rett Association
and the American RSRF for financial support and aid in communication with the families and doctors. We thank Dr Jane Hickey for
the collaboration.
.....................
Petel-Galil, Benteev, Galil, et al
10
11
12
13
14
15
16
Authors’ affiliations
Y Petel-Galil, Y P Galil, I Greenbaum, M Vecsler, B Goldman, Danek
Gertner Institute of Human Genetics, Sheba Medical Center, Tel
Hashomer, Israel
B Benteer, B B Zeev, Child Neurology Department, Sheba Medical
Center, Tel Hashomer, Israel
Y P Galil, M Vecsler, B Goldman, E Gak, Sackler School of Medicine, Tel
Aviv University, Tel Aviv, Israel
H Lohi, B A Minassian, Program in Genetic and Genomic Biology,
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada
B A Minassian, Institute of Medical Sciences, University of Toronto,
Toronto, Ontario, Canada
17
18
19
20
21
Funding: This study was supported by the Central Fund for the
Development of Services for the Retarded in the Local Councils, Israel.
BAM was supported by the Rett Syndrome Research Foundation and HL
was supported by the Sigrid Juselius Foundation, Finland.
22
Competing interests: None declared.
23
This work is part of the requirements of the PhD thesis of YPG at the
Department of Human Genetics, Sackler School of Medicine, Tel Aviv
University, Tel Aviv, Israel.
24
Correspondence to: E Gak, Danek Gertner Institute of Human Genetics,
Sheba Medical Center, Tel Hashomer 52621, Israel; Eva.Gak@sheba.
health.gov.il
Received 26 January 2006
Revised 17 May 2006
Accepted 20 May 2006
25
26
27
28
REFERENCES
1 Hagberg B, Hagberg G. Rett syndrome: epidemiology and geographical
variability. Eur Child Adolesc Psychiatry 1997;6(Suppl 1):5–7.
2 Kerr AM, Stephenson JB. Rett’s syndrome in the west of Scotland. Br Med J (Clin
Res Ed) 1985;291:579–82.
3 Hagberg B. Rett’s syndrome: prevalence and impact on progressive severe
mental retardation in girls. Acta Paediatr Scand 1985;74:405–8.
4 Hagberg B, Hanefeld F, Percy A, Skjeldal O. An update on clinically
applicable diagnostic criteria in Rett syndrome. Comments to Rett Syndrome
Clinical Criteria Consensus Panel Satellite to European Paediatric Neurology
Society Meeting, Baden, Germany, 11 September 2001. Eur J Paediatr
Neurol 2002;6:293–7.
5 Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. Rett
syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpGbinding protein 2. Nat Genet 1999;23:185–8.
6 Naidu S, Bibat G, Kratz L, Kelley RI, Pevsner J, Hoffman E, Cuffari C, Rohde C,
Blue ME, Johnston MV. Clinical variability in Rett syndrome. J Child Neurol
2003;18:662–8.
7 Lee SS, Wan M, Francke U. Spectrum of MECP2 mutations in Rett syndrome.
Brain Dev 2001;23(Suppl 1):S138–43.
8 Kriaucionis S, Bird A. The major form of MeCP2 has a novel N-terminus
generated by alternative splicing. Nucleic Acids Res 2004;32:1818–23.
9 Mnatzakanian GN, Lohi H, Munteanu I, Alfred SE, Yamada T, MacLeod PJ,
Jones JR, Scherer SW, Schanen NC, Friez MJ, Vincent JB, Minassian BA. A
www.jmedgenet.com
29
30
31
32
33
34
35
previously unidentified MECP2 open reading frame defines a new protein
isoform relevant to Rett syndrome. Nat Genet 2004;36:339–41.
Nicolao P, Carella M, Giometto B, Tavolato B, Cattin R, GiovannucciUzielli ML, Vacca M, Regione FD, Piva S, Bortoluzzi S, Gasparini P. DHPLC
analysis of the MECP2 gene in Italian Rett patients. Hum Mutat
2001;18:132–40.
Erlandson A, Samuelsson L, Hagberg B, Kyllerman M, Vujic M, Wahlstrom J.
Multiplex ligation-dependent probe amplification (MLPA) detects large
deletions in the MECP2 gene of Swedish Rett syndrome patients. Genet Test
2003;7:329–32.
Laccone F, Junemann I, Whatley S, Morgan R, Butler R, Huppke P, Ravine D.
Large deletions of the MECP2 gene detected by gene dosage analysis in
patients with Rett syndrome. Hum Mutat 2004;23:234–44.
Tao J, Van Esch H, Hagedorn-Greiwe M, Hoffmann K, Moser B, Raynaud M,
Sperner J, Fryns JP, Schwinger E, Gecz J, Ropers HH, Kalscheuer VM.
Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5/STK9) gene
are associated with severe neurodevelopmental retardation. Am J Hum Genet
2004;75:1149–54.
Weaving LS, Christodoulou J, Williamson SL, Friend KL, McKenzie OL,
Archer H, Evans J, Clarke A, Pelka GJ, Tam PP, Watson C, Lahooti H,
Ellaway CJ, Bennetts B, Leonard H, Gecz J. Mutations of CDKL5 cause a
severe neurodevelopmental disorder with infantile spasms and mental
retardation. Am J Hum Genet 2004;75:1079–93.
Amir RE, Van den Veyver IB, Schultz R, Malicki DM, Tran CQ, Dahle EJ,
Philippi A, Timar L, Percy AK, Motil KJ, Lichtarge O, Smith EO, Glaze DG,
Zoghbi HY. Influence of mutation type and X chromosome inactivation on Rett
syndrome phenotypes. Ann Neurol 2000;47:670–9.
Allen RC, Zoghbi HY, Moseley AB, Rosenblatt HM, Belmont JW. Methylation
of HpaII and HhaI sites near the polymorphic CAG repeat in the human
androgen-receptor gene correlates with X chromosome inactivation. Am J Hum
Genet 1992;51:1229–39.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 22DDC(T) method. Methods 2001;25:402–8.
Yusufzai TM, Wolffe AP. Functional combinations of Rett syndrome mutations
on human MeCP2. Nucleic Acids Res 2000;28:4172–9.
Amano K, Nomura Y, Segawa M, Yamakawa K. Mutational analysis of the
MECP2 gene in Japanese patients with Rett syndrome. J Hum Genet
2000;45:231–6.
Amir RE, Fang P, Yu Z, Glaze DG, Percy AK, Zoghbi HY, Roa BB, Van den
Veyver IB. Mutations in exon 1 of MECP2 are a rare cause of Rett syndrome.
J Med Genet 2005;42:e15.
Fukuda T, Yamashita Y, Nagamitsu S, Miyamoto K, Jin JJ, Ohmori I,
Ohtsuka Y, Kuwajima K, Endo S, Iwai T, Yamagata H, Tabara Y, Miki T,
Matsuishi T, Kondo I. Methyl-CpG binding protein 2 gene (MECP2) variations
in Japanese patients with Rett syndrome: pathological mutations and
polymorphisms. Brain Dev 2005;27:211–17.
Huppke P, Held M, Hanefeld F, Engel W, Laccone F. Influence of mutation
type and location on phenotype in 123 patients with Rett syndrome.
Neuropediatrics 2002;33:63–8.
Smeets E, Schollen E, Moog U, Matthijs G, Herbergs J, Smeets H, Curfs L,
Schrander-Stumpel C, Fryns JP. Rett syndrome in adolescent and adult
females: clinical and molecular genetic findings. Am J Med Genet
2003;122A:227–33.
Schanen C, Houwink EJ, Dorrani N, Lane J, Everett R, Feng A, Cantor RM,
Percy A. Phenotypic manifestations of MECP2 mutations in classical and
atypical Rett syndrome. Am J Med Genet A, 2004;126:129–40.
Kudo S, Nomura Y, Segawa M, Fujita N, Nakao M, Schanen C, Tamura M.
Heterogeneity in residual function of MeCP2 carrying missense mutations in
the methyl CpG binding domain. J Med Genet 2003;40:487–93.
Shahbazian MD, Zoghbi HY. Molecular genetics of Rett syndrome and clinical
spectrum of MECP2 mutations. Curr Opin Neurol 2001;14:171–6.
Young JI, Zoghbi HY. X-chromosome inactivation patterns are unbalanced
and affect the phenotypic outcome in a mouse model of Rett syndrome.
Am J Hum Genet 2004;74:511–20.
Trappe R, Laccone F, Cobilanschi J, Meins M, Huppke P, Hanefeld F, Engel W.
MECP2 mutations in sporadic cases of Rett syndrome are almost exclusively of
paternal origin. Am J Hum Genet 2001;68:1093–101.
Abuhatzira L, Makedonski K, Galil YP, Gak E, Ben Zeev B, Razin A, Shemer R.
Splicing mutation associated with Rett syndrome and an experimental
approach for genetic diagnosis. Hum Genet 2005;118:91–8.
Zhao J, Hyman L, Moore C. Formation of mRNA 39 ends in eukaryotes:
mechanism, regulation and interrelationship with other steps in mRNA
synthesis. Microbiol Mol Biol Rev 1999;63:405–45.
Baker KE, Parker R. Nonsense-mediated mRNA decay: terminating erroneous
gene expression. Curr Opin Cell Biol 2004;16:293–9.
Kaufmann WE, Jarrar MH, Wang JS, Lee YJ, Reddy S, Bibat G, Naidu S.
Histone modifications in Rett syndrome lymphocytes: a preliminary evaluation.
Brain Dev 2005;27:331–9.
Ravn K, Nielsen JB, Uldall P, Hansen FJ, Schwartz M. No correlation between
phenotype and genotype in boys with a truncating MECP2 mutation. J Med
Genet 2003;40:e5.
Van Esch H, Bauters M, Ignatius J, Jansen M, Raynaud M, Hollanders K,
Lugtenberg D, Bienvenu T, Jensen LR, Gecz J, Moraine C, Marynen P, Fryns JP,
Froyen G. Duplication of the MECP2 region is a frequent cause of severe
mental retardation and progressive neurological symptoms in males. Am J Hum
Genet 2005;77:442–53.
Collins AL, Levenson JM, Vilaythong AP, Richman R, Armstrong DL,
Noebels JL, David Sweatt J, Zoghbi HY. Mild over-expression of MeCP2 cause
a progressive neurological disorder in mice. Hum Mol Genet
2004;13:629–39.